Stephen Clum

Cotranslational Membrane Insertion of the Serine Proteinase Precursor NS2B-NS3(Pro) of Dengue Virus Type 2 Is Required for Efficient in Vitro Processing and Is Mediated through the Hydrophobic Regions of NS2B

Polyprotein processing of dengue virus type 2, a positive strand RNA virus, is carried out by the host signal peptidase and a novel two-component viral proteinase of the serine proteinase family, NS2B/NS3(Pro), in the endoplasmic reticulum. Using an in vitro processing system, we examined the cis andtrans cleavages of the 2B/3 and 4B/5 sites by NS2B/NS3(Pro), respectively. Lysates of BHK-21 cells coexpressing NS2B and NS3(Pro) mediated trans cleavage of the 4B/5 sitein vitro, and the protease activity was associated with the membrane fraction. To study the role of membranes in the protease activity of NS2B/NS3(Pro), labeled precursors, NS2B-NS3(Pro), and the mutant ndNS2B-NS3(Pro) in which the functional hydrophilic domain of NS2B was deleted, were analyzed using a coupled in vitrotranscription/translation system (TnT). The results showed that cotranslational addition of microsomal membranes to the TnT reaction markedly enhanced the cis cleavage of the 2B/3 site in a dose-dependent manner. NS2B synthesized in the presence of membranes also facilitated trans cleavage of the 2B/3 site in the mutant precursor. The cleavage products, NS2B and NS3(Pro), were membrane-associated. Furthermore, this membrane requirement was dictated by the hydrophobic regions of NS2B. Deletion of hydrophobic regions of NS2B, leaving only the conserved hydrophilic domain of 40 amino acids, resulted in highly efficient processing of the 2B-3 sitein vitro in the absence of microsomal membranes.

Anticipating, Preventing, and Treating Complications in Patients With Limited Pulmonary Reserve

F iberoptic bronchoscopy as a tool for diagnosis, therapeutic, and palliative intervention in patients with pulmonary pathology was first described in 1967. Published rates of complications from fiberoptic bronchoscopy have ranged from <0.1% to 11%; however, mortality rates are low, estimated at <0.1%. Complications include respiratory failure, laryngospasm, bronchospasm, bleeding, and pneumothorax. Although complication rates are low, these procedures are often indicated in patients with limited pulmonary reserve.1 The article entitled “Bronchoscopy safety in patients with chronic obstructive lung disease (COPD)” by Bellinger et al2 is an important one. They prospectively looked at 258 patients over 12 months who underwent bronchoscopy under moderate sedation as outpatients. Sixty-seven (44%) had COPD, with 6 (9%) having mild, 29 (42%) having moderate, 27 (41%) having severe, and 5 (8%) having very severe disease. Thirteen percent of the COPD patients had minor complications and 5% had severe complications. The severe and very severe COPD patients had significantly more complications compared with the patients without COPD. This study is prospective and well performed. The COPD patients were on inhalers, but the authors did not check whether each patient’s treatment was optimum before the bronchoscopy. This is also a single-center study. Identification of at-risk patient populations for bronchoscopy allows one to anticipate and prevent or reduce procedural complications in high-risk patients. This study helps define those patients at greatest risk for procedural complications. In this article, patients with severe and very severe COPD, defined as those with forced vital capacity in 1 second (FEV1) <50% (severe COPD) and <30% (very severe COPD), experienced the most procedural complications. Understanding of medications and management techniques that can decrease procedural risk or utilization of bronchoscopy techniques for rapid intervention of procedure-related complications is critical for optimal patient management. This includes choice of sedation, interface for oxygen administration during and after the procedure, pharmacological interventions to prevent laryngospasm, monitoring and treatment of bronchospasm, rapid control of procedure-related bleeding, and identification and treatment of procedure-related pneumothorax. Interventions shown to reduce bronchoscopy-related risk and treat procedural complications have been described. Sedation during bronchoscopy can precipitate hypoxic and hypercapnic respiratory failure. Ideal